This invention relates to a biosensor by which the concentration of L-phenylalanine contained in various samples can be quickly and conveniently quantitated without resort to any troublesome pretreatments. More particularly speaking, it relates to a biosensor which is useful in quantitating L-phenylalanine contained in biological samples (blood, urine, saliva, sweat, etc.), food samples and the like by an electrochemical measurement method with the use of L-phenylalanine dehydrogenase, etc. This method is particularly significant in newborn mass screening for detecting phenylketonuria (PKU), which is an amino acid metabolic error, at the early stage, or monitoring the daily living of patients suffering form this disease.
L-Phenylalanine is an important amino acid which is one of the essential amino acids and contained in a large amount of biological samples as well as in foods and drinks employed as L-phenylalanine sources. On the other hand, the disease known as PKU is one of typical hereditary amino acid metabolic errors wherein tyrosine is not synthesized due to L-phenylalanine dehydrogenase deficiency and thus L-phenylalanine is pooled at an abnormally high level in the blood. When allowed to stand, PKU induces serious intellectual disturbance, speech disturbance, amelanotic symptom, etc.
To prevent this disease, newborn mass screening has been widely carried out in Japan and foreign countries. As a typical example of methods for determining L-phenylalanine concentration in the blood, the Guthrie method with the use of dried blood spot has been employed and has largely contributed to early diagnoses.
Patients with PKU thus found should have a diet therapy restricting their L-phenylalanine intake. Namely, such patients should have specially prepared dishes with the elimination or reduction of L-phenylalanine at least until reaching the majority, preferably throughout their lives. Since L-phenylalanine is one of the essential amino acids for the human-body, the L-phenylalanine concentration should be strictly regulated so that it can be taken at the maximum level without inducing brain disturbance, etc. but yet at the minimum level required for the growth of the body. In treating PKU, it is therefore essential to monitor not only the phenylalanine level in the blood but also the phenylalanine intake in the daily diet.
Under these circumstances, examples of methods for determining L-phenylalanine by using blood as samples include the liquid chromatographic method (Journal of Chromatography, Vol. 274, p. 318 (1983)) and the bioassay method with the use of dried blood spot, i.e., the method widely known as the Guthrie method (Pediatrics, Vol. 32, p. 338 (1963)). In the former method it is necessary to subject samples to a specific pretreatment and to use expensive measuring instruments. Although the latter method can be conveniently carried out, it takes a long time to complete the reaction and the results need to be scrutinized with the naked eye. Namely, each of these methods suffers from some problems from the viewpoint of convenience or rapidness in quantification.
There have been also reported determination methods with the use of enzymes, for example, a method with the use of L-phenylalanine ammonia-lyase (Methods of Enzymatic Analysis, Vol. 8, p. 405 (1985)), a method with the use of L-phenylalanine oxidase (Clinica Chimica Acta, Vol. 136, p. 131 (1984)) and a method with the use of L-phenylalanine dehydrogenase (Japanese Laid-Open Patent Publication No. 63-129996(A)). In these documents, the usefulness of these methods in quantification is pointed out. In particular, the method of determining phenylalanine in the blood with the use of L-,phenylalanine dehydrogenase is reported in detail by Hummel et al. (Analytical Biochemistry, Vol. 170, p. 397 (1988)) and Wendel et al. (Analytical Biochemistry, Vol. 180, p. 91 (1989), Clinica Chimica Acta, Vol. 192, p. 165 (1990)). In each of these methods, the blood employed as a,sample is subjected to various pretreatments and then an enzyme reaction is carried out. In addition, the quantification cannot be performed unless expensive and relatively large-scale measuring instruments (a spectrophotometer, etc.) are employed.
In recent years, biosensors, in particular, sensors with the use of enzymes have been vigorously developed. Through them all, it has become possible to conveniently and highly accurately determine blood glucose level as disclosed in EP 351891 and WO 86/07632. Although attempts have been recently made by Huang et al. to determine L-phenylalanine (Analytical Chemistry, Vol. 70, p. 991 (1998)), there still remain a number of problems to be solved in putting the sensor to practical use.
In this method, a carbon paste containing L-phenylalanine dehydrogenase mixed with two other enzymes (i.e., salicylate hydroxylase and tyrosinase) is filled in a tube (1.2 mmxc3x971.5 mmxc3x9730 mm) which is provided with a copper wire as a lead wire. After polishing the electrode surface, it is employed as a working electrode. A detection unit composed of this working electrode, a reference electrode made of silver/silver chloride (Ag/AgCl) and a counter electrode made of platinum is connected to a reactor to thereby fabricate a determination unit. The determination is carried out by mixing a buffer, a salicylic acid solution and oxidized nicotinamide adenine dinucleotide (NAD+) in the reactor, adding a sample, and calculating the L-phenylalanine concentration by using a computer from the response current obtained after the reaction. Thus phenylalanine in a concentration range of from 20 to 150 xcexcM can be quantitated and the detection sensitivity is 5 xcexcM. However, there still remain problems to be solved in, for example, the stability of the response and the storage stability of the enzyme electrode thus constructed.
Although the biosensor technology as described above is just making it possible to determine L-phenylalanine by using L-phenylalanine dehydrogenase, a quick and convenient quantification is still impossible since it is necessary in this method to prepare reagents and instruments and perform troublesome procedures and operations. Although it is desirable for patients with PKU that L-phenylalanine can be conveniently determined at home so as to monitor the blood L-phenylalanine level and examine foods and drinks to be cooked, it is still difficult at the present stage.
Under these circumstances, it is an object of the present invention to provide a biosensor, by which L-phenylalanine can be highly accurately, quickly and conveniently quantitated without resort to the use of many reagents, large-scaled measuring devices or instruments, troublesome pretreatments or special techniques, and a determination method therefor.
To achieve the above-described object, the present invention provides an L-phenylalanine sensor composed of an electrode system, which comprises at least a working electrode and a counter electrode formed on an insulating support, and a reagent reaction layer, which contains as reaction reagents at least L-phenylalanine dehydrogenase, NAD+ or oxidized nicotinamide adenine dinucleotide phosphate (NADP+) as a coenzyme and an electron mediator and is integrated with the above electrode system, and a method of determining L-phenylalanine by which L-phenylalanine. can be electrochemically quantitated simply by adding a sample to the above-described sensor without resort to any troublesome pretreatments.
In the L-phenylalanine sensor according to the present invention, an absorbent carrier containing as reaction reagents at least L-phenylalanine dehydrogenase, NAD+ or NADP+ and an electron mediator is located as a reagent reaction layer between the electrodes of an electrode system, which comprises at least a working electrode and a counter electrode provided opposite to each other on an insulating support, and integrated with the electrode system. Owing to this structure, both of the enzyme reaction and the electrode reaction between the electron mediator and the electrode surface can be carried out and, furthermore, the sensor can be downsized. Use of the absorbent carrier also makes it possible to add a sample smoothly in a definite amount.
The biosensor according to the present invention having the structure as described above makes it possible to conveniently and highly accurately quantitate L-phenylalanine contained in a sample without using any expensive special instruments or adding reagents such as an enzyme to the sample.
According to the present invention, a sample is added to an L-phenylalanine sensor wherein an electrode and a reagent reaction layer are integrated together. Thus, the L-phenylalanine contained in the sample is introduced into the enzyme reaction by the L-phenylalanine dehydrogenase and NAD+ or NADP+. As a result, phenylpyruvate is formed and NAD(P)+ is reduced into NAD(P)H. In association therewith, the electron mediator is reduced due to the electron transfer from NAD(P)H. Subsequently, a potential is applied and thus the reduced electron mediator is oxidized as the electrode reaction, thereby causing the generation of an oxidation current. Thus, L-phenylalanine can be electrochemically quantitated with the guidance of the response current corresponding to the substrate concentration.
The present invention further provides an L-phenylalanine sensor composed of an electrode system, which comprises at least a working electrode and a counter electrode formed on an insulating support, and a reagent reaction layer which contains reaction reagents and is integrated with the above-described electrode system. In this L-phenylalanine sensor, the reagent reaction layer comprises an,absorbent carrier containing at least L-phenylalanine dehydrogenase, NAD+ or NADP+ and an electron mediator and is located in a part where the electrode reaction between these electrode arises, thereby giving an integral structure.
Use of the absorbent carrier in the reagent reaction layer makes it possible to provide a method of conveniently maintaining the reaction reagents while achieving excellent storage stability of the reagents and excellent response stability with neither processing the electrode system nor affecting (for example, worsening the performance) the same. Moreover, a definite amount of a sample can be quickly introduced into the reagent reaction layer thereby. Thus, L-phenylalanine can be highly accurately and conveniently quantitated within a short period of time.